Biochemical Journal

Research article

Processing of proSAAS in neuroendocrine cell lines

Nino MZHAVIA, Yimei QIAN, Yun FENG, Fa-Yun CHE, Lakshmi A. DEVI, Lloyd D. FRICKER

Abstract

ProSAAS, a recently discovered granin-like protein, potently inhibits prohormone convertase (PC)1, and might also perform additional functions. In the present study, the processing of proSAAS was compared in two neuroendocrine cell lines overexpressing this protein: the AtT-20 mouse pituitary corticotrophic line and the PC12 rat adrenal phaeochromocytoma line. The processing of proSAAS was examined by pulse–chase analysis using [3H]leucine, by MS, and by chromatography and radioimmunoassay. Various smaller forms of proSAAS were detected, including peptides designated as little SAAS, PEN and big LEN. Because the PC-12 cells used in the present study do not express either PC1 or PC2, the finding that these cells efficiently cleave proSAAS indicates that these cleavages do not require either enzyme. Two of the peptides identified in AtT-20 media represent novel C-terminally truncated forms of PEN. In both cell lines, the secretion of the small proSAAS-derived peptides is stimulated by secretagogues. However, long-term treatment of wild-type AtT-20 cells with two different secretagogues (8-bromo-cAMP and a phorbol ester) does not affect levels of proSAAS mRNA; this treatment significantly increases PC1 mRNA by approx. 60–80%. The lack of co-regulation of proSAAS and PC1 mRNA implies that enzyme activity can be induced without an accompanying increase in the inhibitor. In addition, the finding that the peptides are secreted via the regulated pathway is consistent with the proposal that they may function as neuropeptides.

  • granin
  • neuropeptide
  • peptide processing
  • prohormone convertase
  • 7B2

Footnotes

  • 1 These authors contributed equally to this study.

  • Abbreviations used: CPE, carboxypeptidase E; DMEM, Dulbecco's modified Eagle's medium; ir, immunoreactive; MALDI–TOF, matrix-assisted laser-desorption ionization–time-of-flight; MS/MS, tandem MS; PACE4, paired basic amino acid converting enzyme 4; PC, prohormone convertase; RIA, radioimmunoassay; RP, reversed phase; TFA, trifluoroacetic acid.